Somitic pax3b is localised to the ventrolateral somite and required for MMP migration. (A) Phylogeny of Pax3 proteins using the Bayesian Monte Carlo Markov chain method with 10,000 bootstrap replicates (MrBayes v3.1.2) (Ronquist and Huelsenbeck, 2003). Tetrapod Pax3 genes are blue; teleost Pax3a and Pax3b clades are green and red, respectively; a cephalochordate Pax3/7 homolog is magenta; and orange indicates insect homologs. (B-N) In situ hybridisation for pax3b (B,C,F-H), pax3a (D,E,I), lbx2 and dlx2a (K-N′). Dorsal view flatmounts have anterior towards the top (B-E) or anterior towards the left (K′-N′). Lateral view wholemounts have anterior towards the left and dorsal towards the top (F,I,K-N′), dorsal view, wholemount has anterior towards the left (G). Transverse cryosection has dorsal towards the top (H). (B,C) Somitic pax3b mRNA was restricted to the anterior somites at 15 and 18 ss (white arrowheads). (D,E) Somitic pax3a was expressed in all somites at 15 and 18 ss. (F-I) At 25 hpf, somitic pax3b was localised to the ventrolateral region of anterior somites (arrowheads, H) and dermomyotome (arrow, H), as was pax3a (I). (J) Somitic pax3b expression dynamics (pink indicates dermomyotome at 25 hpf). (K-N′) Rostral extension of lbx2+ cells (yellow arrowheads) had reached the hyoid arch (dotted outline) at 24 hpf in controls, but was severely reduced in pax3b morphants and pax3a+pax3b double morphants. Scale bars: 100 μm. (O-Q) Rostral migration of lbx2+ cells at 24 hpf was significantly reduced (O; group A versus B; P<0.0001) as was the area of hindbrain lbx2+ expression (brackets in K-N and K′-N′) (P; P<0.0001; groups A-C differ significantly P<0.05). By contrast, the area of dlx2a expression in pharyngeal arches was unaltered after pax3 manipulation (Q; P=0.7282). Data are mean+s.e.m.
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